Lyme disease has emerged as a major public health threat in the US. Currently, no human vaccine is available. Although targeting tick populations with chemical acaricides is an effective way of reducing the risk of infection, the use of acaricides has adverse health effects and raises environmental concerns. A host-targeted approach by deploying doxycycline hyclate-laden baits to the field showed dramatic reduction of infection rates in both rodent reservoirs and Ixodes scapularis ticks by Borrelia burgdorferi, the agent of Lyme disease. However, antibiotics such as doxycycline are not suitable for such purpose as they are used for treating patients. Nevertheless, such proof of concept study demonstrates that a host-targeted approach using alternative compounds is a promising approach to eliminate spirochetes in rodent reservoir hosts and tick vectors. The bacterial second messengers, c-di-GMP and c-di-AMP, have emerged as central regulators for bacterial physiology and are potential drug targets. Many bacteria encode multiple copies of cyclases for the synthesis of c-di-GMP and c-di-AMP, which make it difficult to target the pathways. Borrelia burgdorferi only has a single diguanylate cyclase Rrp1 and a single diadenylate cyclase CdaA for c-di-AMP synthesis, which makes them attractive drug targets. As shown in the preliminary data, we found that these two cyclic dinucleotide, one controls spirochetes? survival in each of the two hosts in B. burgdorferi enzootic cycle, ticks and mammals: while c-di-GMP is essential for tick colonization, c-di-AMP is indispensable for mammalian infection. The hypothesis of this proposal is that small molecule inhibitors targeting c-di-GMP and c-di- AMP cyclases, Rrp1 and CdaA, would eliminate B. burgdorferi in ticks and mammalian reservoirs, which can be exploited to reduce Lyme disease incidence. The co-PI of this proposal, Dr. Herman Sintim, a Drug Discovery Professor of Chemistry, pioneered developing inhibitors for bacterial diguanylate cyclase and diadenylate cyclase, and his group has already reported several potent inhibitors against these cyclases of other bacteria. Accordingly, we propose to develop a strategy to target CdaA of B. burgdorferi to eliminate in spirochetes rodent reservoirs (Aim 1), and a strategy to target Rrp1 of B. burgdorferi to eliminate spirochetes in ticks (Aim 2). We will also test a combination of inhibitors targeting both pathways to eliminate B. burgdorferi in its enzootic cycle. The underlying mechanisms of how c-di-AMP and c-di-GMP are employed by B. burgdorferi to survive in mammals and ticks will also be investigate. This proposal address one of the specific Focus Areas listed in this RFA: reservoir-targeted approaches to interrupt the natural history of infection. Upon accomplishing the proposed work, further field experiments will be conducted using animal baits containing a combination of both inhibitors to evaluate their effects on reducing spirochete burden in ticks and animals in nature. Such host-targeted strategy will have significant impact on combating Lyme disease.